EP0964916B1 - Hbv mutants and methods for detecting same - Google Patents

Hbv mutants and methods for detecting same Download PDF

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Publication number
EP0964916B1
EP0964916B1 EP97934385A EP97934385A EP0964916B1 EP 0964916 B1 EP0964916 B1 EP 0964916B1 EP 97934385 A EP97934385 A EP 97934385A EP 97934385 A EP97934385 A EP 97934385A EP 0964916 B1 EP0964916 B1 EP 0964916B1
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Prior art keywords
hbv
variant
xaa
dna
polymerase
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German (de)
English (en)
French (fr)
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EP0964916A1 (en
EP0964916A4 (en
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Stephen Alister Locarnini
Angeline Ingrid Bartholomeusz
Thein Thein Aye
Robert A. De Man
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Melbourne Health
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Melbourne Health
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates generally to viral variants exhibiting reduced sensitivity to particular agents. More particularly, the present invention is directed to hepatitis B variants exhibiting complete or partial resistance to nucleoside analogues. The present invention further contemplates assays for detecting such viral variants which assays are useful in monitoring anti-viral therapeutic regimes.
  • Xaa 1 nXaa 2 Specific mutations in an amino acid sequence are represented herein as "Xaa 1 nXaa 2 " where Xaa 1 is the original amino acid residue before mutation, n is the residue number and Xaa 2 is the mutant amino acid.
  • the abbreviation "Xaa” may be the three letter or single letter amino acid code.
  • the amino acid residues for Hepatitis B virus DNA polymerase are numbered with the residue methionine in the motif Tyr Met Asp Asp (YMDD) being residue number 550.
  • YMDD residue methionine
  • Australian Patent Application No. PO3519 filed 8 November 1996. the same methionine was designated residue 530.
  • the amino acid residues for the DNA polymerase referred to in this specification have been re-numbered accordingly.
  • HBV Hepatitis B Virus
  • the HBV genome is of a complex nature having a partially double stranded DNA structure with overlapping open reading frames encoding surface, core, polymerase and X genes.
  • the complex nature of the HBV genome is represented in Figure 1.
  • nucleoside analogues could act as effective anti-viral agents.
  • nucleoside analogues currently being tested are penciclovir and its oral form famciclovir (2, 3, 4, 5) and lamivudine (6,7). There is potential for such agents to be used in the treatment of chronic HBV infection.
  • Peniciclovir has been recently shown to have potent inhibitory activity against duck HBV DNA synthesis in vitro and has been shown to inhibit HBV DNA polymerase-reverse transcriptase activity in vitro (8,9). Similarly, oral famiciclovir has been demonstrated to inhibit intra-hepatic replication of duck HBV virus in vivo (10). In man, famciclovir has been shown to reduce HBV DNA replication in a patient with severe hepatitis B following orthotopic liver transplantation (OLT) (11). Penciclovir has been shown to induce mutations in region B of the HBV polymerase (18). 3TC in associated with mutations in the highly conserved YMDD motif of the HBV polymerase (16).
  • nucleoside analogue antiviral therapy was used to control severe post-OLT recurrence of HBV infection (12).
  • Long term therapy is mandatory where patients are immunosuppressed and the rate of HBV replication is very high.
  • any long term chemotherapy of infectious agents there is a potential for development of resistance or reduced sensitivity to the therapeutic agents employed.
  • the inventors have identified variants of HBV with mutations in the HBV DNA polymerase gene which to varying extents reduce the sensitivity of HBV to nucleoside analogues.
  • the identification of these HBV variants is important for the development of assays to monitor nucleoside analogue therapeutic regimes and to screen for agents which can mask the effects of the mutation.
  • the HBV genome comprises a series of overlapping open reading frames, a nucleotide mutation in one open reading frame can affect translation products in another open reading frame.
  • the present invention is directed to a variant of an isolated HBV virus which replicates via an RNA intermediate wherein said variant comprises a nucleotide mutation in a gene encoding a DNA polymerase resulting in at least one amino acid Leu to Met, substitution to said DNA polymerase at position 526.
  • the mutation in the DNA polymerase results in decreased sensitivity of the HBV to a nucleoside analogue.
  • Regions of the HBV polymerase show amino acid similarity with other RNA-dependent DNA polymerases and RNA-dependent polymerases (13). In this specification, reference is made to the conserved regions defined by Poch et al (13) as domains B and C.
  • the mutation results in an altered amino acid sequence in the B domain of the HBV DNA polymerase.
  • an HBV variant comprising a mutation in the nucleotide sequence encoding a DNA polymerase resulting in an amino acid Leu to Met substitution in said DNA polymerase in its B domain at position 526 and wherein said variant exhibits decreased sensitivity to a nucleoside analogue.
  • nucleoside analogues contemplated by the present invention include penciclovir and its oral form famciclovir as well as lamivudine (3TC).
  • the B domain is considered to comprise amino acid residues 505 to 529 of HBV DNA polymerase. This sequence is represented as follows:
  • Reference to the B domain includes reference to proximal regions which includes up to about 20 amino acids on either side of the domain.
  • the mutation is in one or more of the following amino acids:
  • the C domain comprises amino acids 546 to 556 as follows:
  • residue numbering in this specification has been adjusted according to the new numbering system where the methione of YMDD is 550.
  • Reference to the C domain includes proximal regions of up to 20 amino acids either side of the domain.
  • resistance is used in its most general sense and includes total resistance or partial resistance or decreased sensitivity to a nucleoside analogue.
  • the variants are in isolated form such that they have undergone at least one purification step away from naturally occurring body fluid.
  • the variants may be maintained in isolated body fluid or may be in DNA farm.
  • the present invention also contemplates infectious molecular clones comprising the genome or parts thereof from a variant HBV.
  • the mutation in the HBV DNA polymerase is Glu Leu526Met, with an unmutated YMDD motif in the C. domain.
  • the identification of the variant% of the present invention permits the generation of a range of assays to detect such variants.
  • the detection of such variants may be important in identifying resistant variants to determine the appropriate form of chemotherapy and/or to monitor vaccination protocols.
  • another aspect of the present invention contemplates a method for determining the potential for an HBV to exhibit reduced sensitivity to a nucleoside analogue, said method comprising isolating DNA or corresponding mRNA from said HBV and screening for a mutation in the nucleotide sequence encoding HBV DNA polymerase resulting in at least one amino acid Leu to Met substitution, in the B domain of said DNA polymerase at position 526 wherein the presence of such a mutation is an indication of the likelihood of resistance to said nucleoside analogue.
  • the assay determines a mutation resulting in a Leu526Met substitution Glu.
  • the DNA or corresponding RNA may be assayed or alternatively the DNA polymerase or surface antigen may be screened for the mutation.
  • the detection according to this aspect of the invention may be any nucleic acid-based detection means, for example nucleic acid hybridisation techniques or polymerase chain reaction (PCR).
  • the invention further encompasses the use of different assay formats of said nucleic acid-based detection means, including restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), single-strand chain polymorphism (SSCP), amplification and mismatch detection (AMD), interspersed repetitive sequence polymerase chain reaction (IRS-PCR), inverse polymerase chain reaction (iPCR) and reverse transcription polymerase chain reaction (RT-PCR), amongst others.
  • RFLP restriction fragment length polymorphism
  • AFLP amplified fragment length polymorphism
  • SSCP single-strand chain polymorphism
  • ATD amplification and mismatch detection
  • IFS-PCR interspersed repetitive sequence polymerase chain reaction
  • iPCR inverse polymerase chain reaction
  • RT-PCR reverse transcription polymerase chain reaction
  • the present invention extends to a range of immunologically based assays to detect variant HB V DNA polymerase.
  • immunologically based assays are based on antibodies directed to naturally occurring HBV DNA polymerase which do not, or substantially do not, interact with the variant HBV DNA polymerase.
  • antibodies to a variant HBV DNA polymerase are used which do not or substantially do not, interact with naturally occurring HBV DNA polymerase.
  • Monoclonal or polyclonal antibodies may be used although monoclonal antibodies are preferred as they can be produced in large quantity and in a homogenous form.
  • a wide range of immunoassay techniques are available such as described in U.S. Patent Nos. 4,016,043, 4,424,279 and 4,018,653.
  • the detection of amino acid variants of DNA polymerase is conveniently accomplished by reference to the consensus amino acid sequence shown in Figure 4.
  • the polymorphisms shown represent the variations shown in various data bases for active pathogenic HBV strains. Where an HBV variant comprises an amino acid different to what is represented, then such an isolate is considered a putative HBV variant having an altered DNA polymerase activity.
  • another aspect of the present.invention contemplates a method for determining whether an HBV isolate encodes a variant DNA polymerase, said method comprising determining the amino acid sequence of its DNA polymerase at position 526 directly or via a nucleotide sequence and comparing same to the amino acid sequence below: where the presence of a different amino acid from the consensus sequence indicates a putative HBV variant.
  • the present invention further contemplates agents which mask the nucleoside analogue resistance mutation. Such agents will be particularly useful in long term treatment by nucleoside analogues.
  • the agents may be DNA or RNA or proteinaceous or non-proteinaceous chemical molecules. Natural product screening such as from plants, coral and microorganisms is also contemplated as a useful potential source of masking agents.
  • the agents may be in isolated form or in the form of a pharmaceutical composition and may be administered sequentially or simultaneously with the nucleoside analogue.
  • kits for assays for variant HBV may, for example, contain the reagents from PCR or other nucleic acid hybridisation technology or reagents for immunologically based detection techniques.
  • the inventors sequenced the HBV polymerase and X open reading frames from a series of isolates from a patient who received antiviral therapy for almost 4 years following post liver transplant recurrence of HBV infection (Figure 2).
  • the patient male, aged 42 years was transplanted because of end-stage liver failure due to chronic HBV infection.
  • Antiviral treatment was commenced approximately 6 months post-OLT.
  • the patient received intravenous (iv) ganciclovir (GCV; 10 mg/kg/day) in combination with iv foscarnet (PFF; 50-125 mg/kg/day; the dose depending on renal function) (12). This is the treatment of GCV+ PFF described in Figure 2 which lasted for 86 days.
  • Serum samples were routinely collected and stored at -70°C. Informed consent was obtained from the patient to use these samples for research purposes.
  • Figure 2 shows the alanine amino transferase (ALT) and HBV DNA levels over the entire course of antiviral treatment. The 5 samples chosen for additional studies cover a period of almost four years.
  • Patient B was retransplanted for pre-core mutant associated HBV-related allograph loss 14 months after the initial liver transplant.
  • Antiviral treatment with GCV (7.5 mg/kg/day) was given for 10 months and then ceased. This was followed by oral famciclovir therapy given (500 mg 3 times/day).
  • HBV polymerase gene was sequenced from a serum HBV sample taken post-transplantation after 850 days FCV therapy.
  • the regions encompassing the catalytic domains of the HBV polymerase were sequenced from a serum sample pretransplant prior to FCV treatment.
  • This patient is treated with famciclovir in which resistance mutation is selected.
  • Hepatitis B surface antigen HbsAg
  • hepatitis B e antigen HbeAg
  • anti-HBe hepatitis B core antigen
  • HbcAg hepatitis B core antigen specific IgG and IgM
  • hepatitis A specific IgM hepatitis delta antigen and antibody
  • anti-hepatitis C virus antibody were measured using commercially available immunoassays (Abbott Laboratories, North Chicago, IL). Only the HBV markers were positive.
  • Hepatitis B viral DNA levels were measured and quantified using a capture hybridisation assay according to the manufacturer's directions (Digene Diagnostics Inc., Beltsville, MD). This patient was infected with a pre-core HBV mutant pre-OLT (12) and this status did not change post-OLT.
  • Samples of serum (100 ⁇ l) were applied to a 20% w/v sucrose cushion in TNE (20 mmol/L Tris-HCI pH 7.4, 150 mmol/L NaCl 2 1 mmol/L EDTA) and centrifuged at 200,000 g for 3 hr at 10°C using an SW41 rotor in a Beckman Model L8 ultracentrifuge. The pellet was resuspended in 50 mmol/L TRIS-HCl pH 7.5 containing 1.5% v/v Triton-X100, 100 mmol/L Kcl and 0.01% v/v 2-mercaptoethanol and allowed to stand overnight at 4°C.
  • HBV DNA post-OLT Upon commencement of the antiviral treatment strategy GCV+ PFF, the level of HBV DNA post-OLT decreased from over 100,000 pg/ml to 10,800 pg/ml by day 87 ( Figure 2). This reduction in viraemia was associated with clinical, biochemical and histological improvement (12). Maintenance famciclovir therapy (treatment GCV) resulted in fluctuating levels of HBV DNA over the ensuring 359 days with two peaks of HBV DNA observed. The switch to oral famciclovir on day 446 was also associated with a rise in HBV DNA, but this was likely to have been the result of insufficient dosing (FCV[I] in Figure 2) rather than a breakthrough in treatment.
  • FCV[I] insufficient dosing
  • HBV DNA Following dose increase to FCV [II] on day 500, there was a decrease in HBV DNA. However, the level of HBV DNA gradually rose over time from 3,000 pg/ml on day 600 (154 days of famciclovir), to 8,800 pg/ml on day 816 (370 days famciclovir), peaking at 29,000 pg/ml on day 1302 (856 days of famciclovir), then stabilising at around 12,000 pg/ml on day 1329 (883 days of famciclovir). A students test of the DNA levels during the treatment period from days 816 to days 1329, revealed statistically significant rise. There was a 1.5 to 2 fold rise in ALT levels over the same time interval (Figure 2) and no change in clinical status.
  • the X and the polymerase genes ofHBV were sequenced at five time points ( Figure 2). During almost 4 years of the antiviral therapy there were no changes in the X gene compared to the pretreatment sequence. However, there were 5 nt changes detected in the polymerase gene from day 816 and day 1329 samples (Table 1). These changes were detected in separate independent PCR amplifications; furthermore the mutations were detected by sequencing both strands and are therefore unlikely to be the result of PCR generated errors. The nt changes in the polymerase gene were first detected after 816 days of treatment, when the patient had been treated with famciclovir for 370 days.
  • HBV patient A
  • HBV patient B
  • 3TC lamvudine [6,7]
  • HBV patient C-FCV
  • All 3TC resistance mutations which developed during treatment with 3TC is shown as HBV (patient C-3TC).
  • the sequence analysis showed a mutation (Thr-Ser substitution) in the HBV polymerase gene near the C domain but no mutation was initially detected in the YMDD motif.
  • a mutation of Met 550 to Ile in the YMDD motif was detected from HBV isolated 32 days (333 days post treatment) after the HBV containing the Thr-Ser substitution was isolated.

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EP97934385A 1996-11-08 1997-08-15 Hbv mutants and methods for detecting same Expired - Lifetime EP0964916B1 (en)

Applications Claiming Priority (3)

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AUPO351996 1996-11-08
AUPO3519A AUPO351996A0 (en) 1996-11-08 1996-11-08 Viral variants and methods for detecting same
PCT/AU1997/000520 WO1998021317A1 (en) 1996-11-08 1997-08-15 Viral variants and methods for detecting same

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EP0964916A1 EP0964916A1 (en) 1999-12-22
EP0964916A4 EP0964916A4 (en) 2001-09-26
EP0964916B1 true EP0964916B1 (en) 2006-12-20

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EP (1) EP0964916B1 (ja)
JP (4) JP2001503277A (ja)
AT (1) ATE348880T1 (ja)
AU (1) AUPO351996A0 (ja)
CA (1) CA2270178C (ja)
DE (1) DE69737126T2 (ja)
ES (1) ES2279542T3 (ja)
HK (1) HK1027374A1 (ja)
WO (1) WO1998021317A1 (ja)

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* Cited by examiner, † Cited by third party
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ZA973367B (en) 1996-04-19 1997-11-18 Innogenetics Nv Method for typing and detecting HBV.
AUPO351996A0 (en) * 1996-11-08 1996-12-05 Western Health Care Network Viral variants and methods for detecting same
USRE40233E1 (en) 1996-11-08 2008-04-08 Melbourne Health Viral variants and methods for detecting same
AUPP706098A0 (en) * 1998-11-11 1998-12-03 North Western Health Care Network Biological compositions, components thereof and uses therefor
AUPP967999A0 (en) * 1999-04-09 1999-05-06 North Western Health Care Network Viral variants
AR028149A1 (es) * 1999-07-08 2003-04-30 Innogenetics Nv Deteccion de la resistencia a los farmacos contra la hepatitis b
AUPQ810900A0 (en) 2000-06-09 2000-07-06 Austin and Repatriation Medical Centre, The Viral variants and methods of using same
SG90149A1 (en) * 2000-07-18 2002-07-23 Government Of The Republic Of Diagnostic assay
US6697858B1 (en) * 2000-08-14 2004-02-24 Telephony@Work Call center
JP4502642B2 (ja) * 2002-02-07 2010-07-14 メルボルン ヘルス ヌクレオシドアナログに対する感受性の変化を有するウイルス変種およびその使用
US7405039B2 (en) 2002-02-07 2008-07-29 Austin Health Viral variants with altered susceptibility to nucleoside analogs and uses thereof
WO2003083094A1 (en) * 2002-03-29 2003-10-09 Innogenetics N.V. Hbv drug resistance drug resistance detection methods
WO2003087351A1 (en) 2002-04-12 2003-10-23 Melbourne Health Hepatitis b viral variants with redused susceptibility to nucleoside analogs and uses thereof
AU2003213874B2 (en) * 2002-04-12 2008-12-04 Austin Health Hepatitis B viral variants with redused susceptibility to nucleoside analogs and uses thereof
AU2003253651C1 (en) 2002-06-14 2010-06-03 Gen-Probe Incorporated Compositions and methods for detecting hepatitis B virus
AU2003277208A1 (en) * 2002-10-01 2004-04-23 Georgetown University Diagnostic for long term response of hbv carrier to 3tc therapy
CN101203528A (zh) 2005-03-15 2008-06-18 基因创新有限公司 对核苷类似物敏感性降低的乙型肝炎病毒变异体及其用途
ES2470318T3 (es) 2005-03-15 2014-06-23 Rheinische Friedrich-Wilhelms-Universit�T Bonn Variantes del virus de la hepatitis B resistentes a ciertos análogos nucle�sidos, pero sensibles a otros, y usos de las mismas
WO2006105597A1 (en) 2005-04-08 2006-10-12 Melbourne Health Variants of hepatitis b virus with resistance to anti-viral nucleoside agents and applications thereof
AU2006230802A1 (en) 2005-04-08 2006-10-12 Austin Health Variants of hepatitis B virus with resistance to anti-viral nucleoside agents and applications thereof
KR100790226B1 (ko) 2005-11-03 2008-01-02 연세대학교 산학협력단 독성 간염 유발 감수성을 검출하는 방법
US20230148447A9 (en) 2008-12-11 2023-05-11 Pacific Biosciences Of California, Inc. Classification of nucleic acid templates
US9175338B2 (en) 2008-12-11 2015-11-03 Pacific Biosciences Of California, Inc. Methods for identifying nucleic acid modifications
WO2010068289A2 (en) * 2008-12-11 2010-06-17 Pacific Biosciences Of California, Inc. Classification of nucleic acid templates
US9611510B2 (en) 2011-04-06 2017-04-04 The University Of Chicago Composition and methods related to modification of 5-methylcytosine (5-mC)
US9238836B2 (en) 2012-03-30 2016-01-19 Pacific Biosciences Of California, Inc. Methods and compositions for sequencing modified nucleic acids
WO2013163207A1 (en) 2012-04-24 2013-10-31 Pacific Biosciences Of California, Inc. Identification of 5-methyl-c in nucleic acid templates
KR20220143812A (ko) * 2019-12-07 2022-10-25 아이에스에이 파마슈티컬즈 비.브이. B형 간염 바이러스와 관련된 질병의 치료

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US5595739A (en) * 1993-05-07 1997-01-21 Abbott Laboratories Hepatitis B virus mutants, reagents and methods for detection
ZA973367B (en) * 1996-04-19 1997-11-18 Innogenetics Nv Method for typing and detecting HBV.
AUPO351996A0 (en) * 1996-11-08 1996-12-05 Western Health Care Network Viral variants and methods for detecting same
USRE40233E1 (en) * 1996-11-08 2008-04-08 Melbourne Health Viral variants and methods for detecting same
AUPP967999A0 (en) * 1999-04-09 1999-05-06 North Western Health Care Network Viral variants
AUPQ810900A0 (en) * 2000-06-09 2000-07-06 Austin and Repatriation Medical Centre, The Viral variants and methods of using same
US7405039B2 (en) * 2002-02-07 2008-07-29 Austin Health Viral variants with altered susceptibility to nucleoside analogs and uses thereof
WO2003083094A1 (en) * 2002-03-29 2003-10-09 Innogenetics N.V. Hbv drug resistance drug resistance detection methods
WO2003087351A1 (en) * 2002-04-12 2003-10-23 Melbourne Health Hepatitis b viral variants with redused susceptibility to nucleoside analogs and uses thereof
JP2007509614A (ja) * 2003-10-21 2007-04-19 メルボルン ヘルス Ｈｂｖ変種の検出および適用
ES2470318T3 (es) * 2005-03-15 2014-06-23 Rheinische Friedrich-Wilhelms-Universit�T Bonn Variantes del virus de la hepatitis B resistentes a ciertos análogos nucle�sidos, pero sensibles a otros, y usos de las mismas
CN101203528A (zh) * 2005-03-15 2008-06-18 基因创新有限公司 对核苷类似物敏感性降低的乙型肝炎病毒变异体及其用途
WO2006105597A1 (en) * 2005-04-08 2006-10-12 Melbourne Health Variants of hepatitis b virus with resistance to anti-viral nucleoside agents and applications thereof
AU2006230802A1 (en) * 2005-04-08 2006-10-12 Austin Health Variants of hepatitis B virus with resistance to anti-viral nucleoside agents and applications thereof
CN101548014B (zh) * 2006-06-06 2012-07-04 墨尔本保健公司 抗病毒抗性突变的检测和应用

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DE69737126T2 (de) 2007-10-04
WO1998021317A1 (en) 1998-05-22
CA2270178C (en) 2012-06-12
US6555311B1 (en) 2003-04-29
JP2012055322A (ja) 2012-03-22
AUPO351996A0 (en) 1996-12-05
EP0964916A1 (en) 1999-12-22
EP0964916A4 (en) 2001-09-26
DE69737126D1 (de) 2007-02-01
JP2015107127A (ja) 2015-06-11
HK1027374A1 (en) 2001-01-12
ATE348880T1 (de) 2007-01-15
CA2270178A1 (en) 1998-05-22
JP2008154590A (ja) 2008-07-10
US20130171620A1 (en) 2013-07-04
US20030124096A1 (en) 2003-07-03
US20100203506A1 (en) 2010-08-12
JP2001503277A (ja) 2001-03-13

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